Drug permeability screening has become a very important tool in the drug development process. Parallel Artificial Membrane Permeability Assay (PAMPA) has become a widely accepted high throughput drug permeability screening method.
In a typical PAMPA format, a pair of multi-well plates are used: a filter plate and a receiver plate. The filter plate includes open wells with a porous filter membrane extending across a bottom end of each well. The filter membrane is typically of polyvinylidine difluoride (PVrDF) or a polycarbonate material. The receiver plate is a typical multi-well plate having closed bottom ends.
Referring to FIG. 1A, a conventional method used to prepare a filter membrane for PAMPA is shown, This prior method involves impregnating the membrane with an alkane solution of lipids. For example, as discussed in U.S. Published Application No. 20033/0219716 A1, published on Nov. 27, 2003, the alkane solution is typically a solution of phospholipids (e.g., 2% Dioleoyl-sn-glycero-3-phosphocholine (DOPC)) in dodecane. Once the filter membranes are prepared, buffered solutions containing the compounds being analyzed are disposed into the wells of the receiver plate. Buffered solutions without the analyzed compounds are disposed into the wells of the filter plate. The filter plate is placed atop the receiver plate with the filter membranes coming into contact with the buffered solutions of compounds disposed in the wells of the receiver plate. The concentrations of the compounds in the solutions of both the receiver plate and the filter plate are analyzed to observe the diffusion of the compounds through the filter membranes.
It has been found that, using prior art techniques, screening experiments must be conducted relatively soon after preparation of filter membranes for PAMPA because filter membranes impregnated with an alkane solution of lipids are unstable. For example, with reference to FIG. 1B, permeabilities measured by PAMPA using fresh prior art filter membranes (used immediately after preparation) and one-day old prior art filter membranes (stored at room temperature) are shown for seven different drug compounds. The filter membranes were impregnated with a 2% solution of phospholipids in dodecane. Significant variations in measured permeabilities were noted, with severe degradation in reliability with the lapse of relatively short periods of time (e.g., one day). Generally, the permeability results increased with time, indicating that the membranes degraded and became more permeable to all the compounds. As a result, filter membranes for PAMPA prepared with prior art techniques are not well-suited to be prepared in advance of testing and stored. The measurements were Carried out with phosphate buffered saline (PBS) as the working buffer.
Therefore, there is a need in the art for stable, precoated filter membranes for PAMPA that can be prepared in advance of drug permeability screening and stored.
Furthermore, the permeability screening of drug candidates using the prior art is challenged by the incorrect prediction of a group of commercial compounds that are classified by the biopharmaceutical classification system (BCS) as high permeability compounds. Examples of these compounds include caffeine, antipyrine, ketoprofen, metoprolol, naproxen, phenytoin, timolol, and theophyline. The BCS defines highly permeable compounds as those that have human oral absorption greater than 90%. These compounds all have human oral absorption greater than 90%. However, the PAMPA permeability values found for these compounds by the prior art are low.
Therefore, there is a need in the art for improving the predictability of the permeability measurement for the currently under-predicted compounds.
Another challenge in the permeability screening of drug candidates using the prior art is from “sticky” compounds—compounds that are likely to bind to the plastic surface of the plate and/or be trapped inside the artificial membrane. “Sticky” compounds may have high mass retention (the percentage of the total mass of the compound lost during the permeability measurement as a result of binding to the plastic surface and/or retaining in the filter membrane). With high mass retention, it is difficult to obtain reliable, quantitative permeability results.
A further challenge in the permeability screening of drug candidates using the prior art is from low solubility compounds. Low solubility compounds precipitate when the dimethyl sulfoxide (DMSO) stock solution of the compound is diluted into the working buffer (usually PBS or other aqueous buffer). This results in difficulty in measuring the concentration of these compounds in the buffer and, therefore, the difficulty in obtaining reliable, quantitative permeability results.
Therefore, there is a need in the art for improving permeability measurements for “sticky” compounds and low solubility compounds.